A significant amount of the space between the cells in the monolayer is occupied by a complex network of proteins and polysaccharides which is referred to as an extracellular matrix (ECM). The ECM consists of two subgroups of molecules: extracellular matrix molecules (ECM molecules) and cell adhesion molecules (CAMs). ECM molecules include collagen, proteoglycans and non-collagen glycoproteins, and CAMs are comprised of the immunoglobulin superfamily of CAMs (IgCAMs), cadherins, selectins, and integrins. The function of the ECM molecules were thought to have been purely structural due to their large presence in connective tissue, but the ECM appears to play a role in survival, development, migration, proliferation and function of the cells immediately surrounding it.
To maintain proliferating cells in culture for an extended period of time, it is necessary to subculture the cells (remove them from one culture vessel, and place them in a new culture vessel containing fresh medium and a lower cell concentration). Each time a population of cells is subcultured, it is referred to as a passage. In order to move the cells from the first culture vessel to the second culture vessel, the monolayer of cells have to be detached from the surface of the first culture vessel, and then dissociated into a single cell suspension. Then, the single cell suspension can be transferred to the fresh culture vessel. Single cells are necessary for basic biological research. For example, cell sorting methods are used to determine the composition of heterogeneous cell populations, and to isolate specific subpopulations of cells with desirable characteristics which can then be used to conduct further research, or used therapeutically in a clinical setting. Cell sorting methods can only be used effectively on single cells. The generation of a single cell suspension also has applications in other areas such as the production of biomolecules and clinical diagnostics.
In addition to isolating cells from primary tissues and subculturing cells in existing cultures, the generation of single cell suspensions is extremely important for a variety of applications. For example, during cell therapy, single cells are delivered to certain sites in order to treat specific conditions. Transplanting aggregates is undesirable because (i) aggregates can plug the delivery device (ii) it is difficult to estimate the number of actual cells that are delivered (iii) cells in aggregates are more susceptible to cell death due to the nutrient and oxygen mass transfer limitations that they suffer and (iv) aggregates are less likely to migrate to areas of damage, respond to local cues, and integrate into the host cellular architecture. Single cells are also necessary for basic biological research. For example, cell sorting methods are used to determine the composition of heterogeneous cell populations, and to isolate specific subpopulations of cells with desirable characteristics which can then be used to conduct further research, or used therapeutically in a clinical setting. Cell sorting methods can only be used effectively on single cells. The generation of a single cell suspension also has applications in other areas such as the production of biomolecules and clinical diagnostics.
Several methods have been developed to generate single cell suspensions from primary tissues, attached cells in culture, and aggregates in culture. These methods involve the use of physical forces (mechanical dissociation), enzymes (enzymatic dissociation), or a combination of both. Mechanical means of detaching cells that are attached to a surface include the use of cell scrapers. Mechanical means of separating cells which are attached to one another include trituration through a narrow bore pipette, fine needle aspiration, vortex disaggregation, and forced filtration through a fine nylon or stainless steel mesh. Whereas all of these methods are effective in creating single cell suspensions, the excessive physical forces involved often result in a significant amount of cell death and cell damage. In situations where the generation of a suspension of viable single cells is the ultimate goal, cell death and cell damage are extremely undesirable. Moreover the manual nature of certain mechanical dissociation protocols (e.g., trituration, which is done by hand) often make it difficult compare measured values (such as cell viability) from different sources since dissociation efficiency varies between individuals. In fact, the manual nature of this procedure may contribute to differences in the physical attributes (e.g., cell concentration, cell viability, cell size distribution etc.) between two otherwise identical samples.
In an attempt to avoid the negative consequences of mechanical dissociation, researchers have used enzymes (either alone or in combination) which are directed towards one or more components in the ECM, also known as passage enzymes. Certain enzymes are known to target and cleave specific molecules present within the ECM. For example, the enzyme trypsin (which cleaves polypeptide chains on the carboxyl side of arginine and lysine residues) is commonly used to detach and dissociate monolayer cultures, whereas collagenase is often used to dissociate primary tissues and aggregates. However, not all cell types can be easily dissociated using enzymes. For those cell types that are susceptible to enzymatic dissociation, it has been shown that enzymes can be detrimental to the cells and negatively impact the ability of the generated single cells to subsequently survive and or divide. For example, when neural stem cell (NSC) aggregates were dissociated using trypsin, the growth rate of the single cells in subsequent culture was found to have been adversely affected relative to single cells generated using mechanical dissociation. This result may be attributable to the fact that trypsin is known to cleave certain classes of cell surface transmitter receptors. In the extreme, enzymes can completely destroy cells. For example, collagenase has been shown to reduce viable cells to debris when used to dissociate neural stem cell aggregates.
Embryonic stem cells (ESCs) are primitive, undifferentiated cells obtained from the inner cell mass of blastocysts prior to the implantation stage in the mammalian uterine endometrium. These undifferentiated cells are deemed pluripotent since they have the ability to differentiate and yield many types of cells. For ESCs to remain undifferentiated in vitro, they need to attach to a substrate, or else the cells aggregate in suspension to form embryoid bodies and start to differentiate. The culture used to grow ESCs provides a surface for the cells to attach. Currently hESCs are propagated as clusters. These clusters need to be manually cleaned and selected with every passage. Due to the difficulty in manipulating the cell clusters, the clusters are exposed to dislodging enzymes, such as trypsin or collagenase, after the initial passages performed via mechanical dissociation. These enzymes significantly increase the level of cell death.
Human embryonic neural stem cells inoculated into serum free medium can be induced to divide and form aggregates over time. Visually, the aggregates contain a significantly greater amount of extracellular matrix compared to embryonic neural stem cell aggregates derived from mice. Currently, the state-of-the-art method of generating a single cell suspension from these aggregates involves mechanical dissociation. However, due to the large quantities of extracellular matrix, mechanical dissociation of human neurosphere aggregates results in a much greater cell death relative to that caused during the mechanical dissociation of murine neural stem cell aggregates. Even in the hands of an experienced researcher, it is not unusual to obtain measured cell viabilities of 50% or less.
Pancreatic stem cells are cells that are believed to give rise to all of the different endocrine tissues within the pancreas. It is anticipated that research efforts that are presently underway using these cells will eventually lead to cell therapy aimed at eliminating Type I diabetes, a currently incurable disease afflicting millions of individuals. At present, due to the prevalence of this disease, and the associated economic impact, there is an extensive amount of research being conducted in an effort to expand this stem cell population. Pancreatic stem cells are obtained from whole pancreatic tissue through a series of fractionations. The fraction containing the stem cells is isolated from the other fractions and placed into a serum free medium. Currently, there are no methods available to expand these cells in vitro. Rather, the medium simply serves to maintain the cells in culture, and delay cell death. The cells in this fraction, including the stem cells are present as large aggregates of primary tissue. Large aggregates are undesirable since cells rapidly begin to die due to nutrient and oxygen limitations. Thus, in order to ensure that the cells survive, and to isolate the stem cells from the rest of the cells, it is necessary to dissociate the tissue into a single cell suspension. At present, there are no reliable or reproducible methods to accomplish this. Until now, the best method utilized by researchers, and the current accepted practice in this field has been to mechanically dissociate the aggregates. However, this method does not result in the generation of a single cell suspension. Rather, many cell aggregates remain. Significantly increasing the intensity and duration of the mechanical dissociation process does not remove these aggregates, but rather, results in the death of large numbers of otherwise viable cells. Thus, despite being the most commonly used procedure in this field, mechanical dissociation is not ideal.
In light of the problems with prior art methods of creating single cell suspensions of viable pluripotent hESCs, a need exists for a new approach that increases the efficiency and ease of culturing hESCs and reduces the negative results of mechanical and enzymatic dissociation.